Microbial humic soil enhancements

ABSTRACT

A method enhances soil by preparing a microbial solution with microbes, a growth medium, and water; iteratively and selectively breeding generations of microbes to arrive at a predetermined microbial solution in a concentrated form of at least 1×107 cfu/ml (colony-forming units per milliliter); adding humic acid with amino acids and protein to support an active microbial population to support active and healthy plant growth; and storing the microbial solution as a solid for enriching the soil with micronutrients, microbial cultures and organic materials.

BACKGROUND

The present invention relates to microbial enhancements for soilenhancement.

Humic acids are a principal component of humic substances, which are themajor organic constituents of soil (humus), peat and coal. It is also amajor organic constituent of many upland streams, dystrophic lakes, andocean water. It is produced by biodegradation of dead organic matter. Itis not a single acid; rather, it is a complex mixture of many differentacids containing carboxyl and phenolate groups so that the mixturebehaves functionally as a dibasic acid or, occasionally, as a tribasicacid. Humic acids can form complexes with ions that are commonly foundin the environment creating humic colloids. Humic acids are insoluble inwater at acid pH, whereas fulvic acids are also derived from humicsubstances but are soluble in water across the full range of pH. Humicand fulvic acids are commonly used as a soil supplement in agriculture,and less commonly as a human nutritional supplement. As a nutritionsupplement, fulvic acid can be found in a liquid form as a component ofmineral colloids. Fulvic acids are poly-electrolytes and are uniquecolloids that diffuse easily through membranes whereas all othercolloids do not.

In a parallel trend, bacterial agricultural microbials are helpful tothe crops in a way that they detoxify the soil and fight the rootdiseases and provide stability to the soil system. They help in nitrogenfixation, phosphate solubilization, iron sequestration, and phytohormonelevel modulation in crops. Due to these factors, the bacterial segmentdominates the agricultural microbials market.

SUMMARY OF THE INVENTION

In one aspect, a method enhances soil by preparing a microbial solutionwith microbes, a growth medium; iteratively and selectively breedinggenerations of microbes to arrive at a predetermined microbial solutionin a concentrated form of at least 1×10⁷ cfu/ml (colony-forming unitsper milliliter); and storing the microbial solution in a container forenriching the soil with micronutrients, microbial cultures and organicmaterials.

In another aspect, an apparatus for enhancing soil includes a tank for amicrobial solution with microbes, a growth medium; a sequencer toiteratively and selectively breeding generations of microbes to arriveat a predetermined microbial solution in a highly concentrated form ofat least 1×10⁷ cfu/ml (colony-forming units per milliliter); and a pumpto dispense the microbial solution into a container to enrich the soilwith micronutrients, microbial cultures and organic materials.

Implementations of the above aspects may include one or more of thefollowing. The microbes can be selected from Bacillus (B.) acidiceler,B. acidicola, B. acidiproducens, B. acidocaldarius, B. acidoterrestrisr,B. aeolius, B. aerius, B. aerophilus, B. agaradhaerens, B. agri, B.aidingensis, B. akibai, B. alcalophilus, B. algicola, B. alginolyticus,B. alkalidiazotrophicus, B. alkalinitrilicus, B. alkalisediminis, B.alkalitelluris, B. altitudinis, B. alveayuensis, B. alvei, B.amyloliquefaciens, B. a. subsp. Amyl, aoliquefaciens, B. a. subsp.plantarum, B. amylolyticus, B. andreesenii, B. aneurinilyticus, B.anthracia, B. aquimaris, B. arenosi, B. arseniciselenatis, B. arsenicus,B. aurantiacus, B. arvi, B. aryabhattai, B. asahii, B. atrophaeus, B.axarquiensis, B. azotofixans, B. azotoformans, B. badius, B. barbaricus,B. bataviensis, B. beijingensis, B. benzoevorans, B. beringensis, B.berkeleyi, B. beveridgei, B. bogoriensis, B. boroniphilus, B.borstelensis, B. brevis Migula, B. butanolivorans, B. canaveralius, B.carboniphilus, B. cecembensis, B. cellulosilyticus, B. centrosporus, B.cereus, B. chagannorensis, B. chitinolyticus, B. chondroitinus, B.choshinensis, B. chungangensis, B. cibi, B. circulans, B. clarkii, B.clausii, B. coagulans, B. coahuilensis, B. cohnii, B. composti, B.curdlanolyticus, B. cycloheptanicus, B. cytotoxicus, B. daliensis, B.decisifrondis, B. decolorationis, B. deserti, B. dipsosauri, B.drentensis, B. edaphicus, B. ehimensis, B. eiseniae, B. enclensis, B.endophyticus, B. endoradicis, B. farraginis, B. fastidiosus, B.fengqiuensis, B. firmus, B. flexus, B. foraminis, B. fordii, B.formosus, B. fortis, B. fumarioli, B. funiculus, B. fusiformis, B.galactophilus, B. galactosidilyticus, B. galliciensis, B. gelatini, B.gibsonii, B. ginsengi, B. ginsengihumi, B. ginsengisoli, B. globisporus,B. g. subsp. globisporus, B. g. subsp. marinus, B. glucanolyticus, B.gordonae, B. gottheilii, B. graminis, B. halmapalus, B.haloalkaliphilus, B. halochares, B. halodenitrificans, B. halodurans, B.halophilus, B. halosaccharovorans, B. hemicellulosilyticus, B.hemicentroti, B. herbersteinensis, B. horikoshii, B. horneckiae, B.horti, B. huizhouensis, B. humi, B. hwajinpoensis, B. idriensis, B.indicus, B. infantis, B. infernus, B. insolitus, B. invictae, B.iranensis, B. isabeliae, B. isronensis, B. jeotgali, B. kaustophilus, B.kobensis, B. kochii, B. kokeshiiformis, B. koreensis, B. korlensis, B.kribbensis, B. krulwichiae, B. laevolacticus, B. larvae, B.laterosporus, B. lautus, B. lehensis, B. lentimorbus, B. lentus, B.licheniformis, B. ligniniphilus, B. litoralis, B. locisalis, B.luciferensis, B. luteolus, B. luteus, B. macauensis, B. macerans, B.macquariensis, B. macyae, B. malacitensis, B. mannanilyticus, B.marisflavi, B. marismortui, B. marmarensis, B. massiliensis, B.megaterium, B. mesonae, B. methanolicus, B. methylotrophicus, B.migulanus, B. mojavensis, B. mucilaginosus, B. muralis, B. murimartini,B. mycoides, B. naganoensis, B. nanhaiensis, B. nanhaiisediminis, B.nealsonii, B. neidei, B. neizhouensis, B. niabensis, B. niacini, B.novalis, B. oceanisediminis, B. odysseyi, B. okhensis, B. okuhidensis,B. oleronius, B. oryzaecorticis, B. oshimensis, B. pabuli, B.pakistanensis, B. pallidus, B. pallidus, B. panacisoli, B. panaciterrae,B. pantothenticus, B. parabrevis, B. paraflexus, B. pasteurii, B.patagoniensis, B. peoriae, B. persepolensis, B. persicus, B. pervagus,B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae,B. pseudalcalophilus, B. pseudofirmus, B. pseudomycoides, B.psychrodurans, B. psychrophilus, B. psychrosaccharolyticus, B.psychrotolerans, B. pulvifaciens, B. pumilus, B. purgationiresistens, B.pycnus, B. qingdaonensis, B. qingshengii, B. reuszeri, B. rhizosphaerae,B. rigui, B. ruris, B. safensis, B. salarius, B. salexigens, B.saliphilus, B. schlegelii, B. sediminis, B. selenatarsenatis, B.selenitireducens, B. seohaeanensis, B. shacheensis, B. shackletonii, B.siamensis, B. silvestris, B. simplex, B. siralis, B. smithii, B. soli,B. solimangrovi, B. solisalsi, B. songklensis, B. sonorensis, B.sphaericus, B. sporothermodurans, B. stearothermophilus, B.stratosphericus, B. subterraneus, B. subtilis, B. s. subsp. inaquosorum,B. s. subsp. spizizenii, B. s. subsp. subtilis, B. taeanensis, B.tequilensis, B. thermantarcticus, B. thermoaerophilus, B.thermoamylovorans, B. thermocatenulatus, B. thermocloacae, B.thermocopriae, B. thermodenitrificans, B. thermoglucosidasius, B.thermolactis, B. thermoleovorans, B. thermophilus, B. thermoruber, B.thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis,B. tianshenii, B. trypoxylicola, B. tusciae, B. validus, B.vallismortis, B. vedderi, B. velezensis, B. vietnamensis, B. vireti, B.vulcani, B. wakoensis, B. weihenstephanensis, B. xiamenensis, B.xiaoxiensis, and B. zhanjiangensis. With a member of Bacillus as themicrobe, the process can use a carrier from one of: liquid, water, dryhumic acid, wet humic acid, urea, soil wetting aid or a penetrant.

In one embodiment, the penetrant can be about 20% alcohol ethoxylate andabout 80% orange oil. Alternatively, surfactants can be added. Thepenetrant can have one or more high terpene (50% by weight or more)based oils, one or more stabilizers, one or more chelating agents, oneor more preservatives, one or more acidic pH adjusters and one or moreorganic solvents.

The microbes can be: Bacillus amyloliquefaciens at 5.85×107^7 cfu/ml,Bacillus lichniformis at 1.80×107^7 cfu/ml, Bacillus pumilus at4.05×107^7 cfu/ml, or Bacillus subtilis at 6.30×107^7 cfu/ml. Leonarditeand urea and water can be used with the microbes.Polyloxy-(1,2-Ethanedily), Alpha-(nonylphenyl)-omega-hydroxy can be usedwith the microbes. The solution can also include Leonardite and water.The microbial solutions can be applied through spraying, wetting,dipping, misting, drenching, showering, fogging, soaking, dampening,drizzling, dousing and splashing.

Advantages of the solutions may include one or more of the following.Soil enrichment solutions of the system stimulate plant growth,rejuvenate the soil, and promote the growth of beneficial soilmicroorganisms. Some embodiments also provide natural pathogens for theprevention, control and/or cure of turf and plant diseases and otherpurposes encouraging germination and/or growth. The solutions containmicroorganism spores and/or colonies that remain viable for at leastabout a year when stored at room temperature. The solutions provide soilenrichment solutions containing viable microorganism spores and/orcolonies, particularly those useful for enriching poor, disturbed soilsor soils having little or no microbial activity because of the heavypast use of chemicals and/or fertilizers. The systems provide solutionscontaining viable micro organism spores and/or colonies of beneficialfungicides that can be used for seed, turf, and leaf treatment for theprevention, control, and/or cure of turf and plant diseases and otherbeneficial purposes. The solutions also provide soil enrichmentsolutions containing microorganism spores and/or colonies that remain atleast about 90% viable for up to at least about 12, preferably 18 monthsat room temperature, i.e., about 20° to 25° C.

These and other advantages are achieved by the present invention, whichprovides a method of preserving and solutions containing microbialspores and/or colonies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary process to selectively breed the microbes foragricultural use.

FIG. 2 shows an exemplary process to produce the microbial products.

FIGS. 3A-3B show exemplary antifungal activity express by differentBacillus spp. strains.

FIG. 4 shows exemplary cellulolytic enzymes synthesized by thebiological control agent which can be involved in two plant defensemechanism against phyto-pathogenic fungi.

FIG. 5 shows exemplary soil enhancement enzyme profiles isolationstandards.

DETAILED DESCRIPTION OF THE INVENTION

A selectively bred microbial solution is disclosed with multiple singlemicrobial series separately cultivated and followed with crosscultivation among those microbial series in a specific sequence andcontains each of those microbial series, and by-products produced bythose crossly cultivated microbial series are used for applications inmodifying soil quality, activating soil, effectively degrading soilpollution, and helping growth of crops in a soil enhancement embodiment.After the selective breeding through the fermentation, the selectivelybred naturally-occurring microorganisms have the ability to penetratethrough the soil while enriching with micronutrients, microbial culturesand organic materials in a highly concentrated stage.

FIG. 1 shows an exemplary process to selectively breed the microbes foragricultural use. First, fermentation media are prepared with a nutrientsupply (1). The nutrients can include a carbon source Dextrose orGlucose. Additional carbon sources can be used with the dextrose orglucose singly or in combination. For example, another carbon source canbe sucrose, for example. Next, a nitrogen source is provide such as soyprotein that has not been genetically modified (2). Next, in (3),micronutrients—Calcium, Magnesium and Zinc are provided. A person ofordinary skilled in the art appreciates that various compositions of thefermentation media can be prepared so long as the nutrients, one or moreof the carbon sources, and the micronutrients are included.

In (4), the fermentation media is prepared using water supply andsterilized using stream sterilizer at 120 degrees Celsius for 45minutes, but the temperature and time can be varied in accordance withtank volume. In (5), the process produces the microbial products, as isdetailed in FIG. 2. At each stage, quality control methods are appliedusing standard plate count method for Shigella, E. Coli, SalmonellaYersinia and Psuedomonas beroginosa for their absence. All products aremanufactured according to USEPA (United States Environmental ProtectionAgency) standards.

The microbes can be: Bacillus amyloliquefaciens at 5.85×107^7 cfu/ml,Bacillus lichniformis at 1.80×107^7 cfu/ml, Bacillus pumilus at4.05×107^7 cfu/ml, or Bacillus subtilis at 6.30×107^7 cfu/ml. Leonarditeand urea and water can be used with the microbes.

Polyloxy-(1,2-Ethanedily), Alpha-(nonylphenyl)-omega-hydroxy can be usedwith the microbes. The solution can also include Leonardite and water.

The Microbial Strain selection and profile of microbial genes arecarefully selected to form the formulation of products. Through strainselections, screening and improvement, the system generates variousbio-fertilizer products for rejuvenating soil and promote plant growth.For example, Bacillus Subtilus has 4,100 genes. These genes each containapproximately 2000 traits. Each one of these traits and its mutation hasover 1000 profile and sub-profile.

With a member of Bacillus as the microbe, the process can include acarrier from one of: liquid, water, dry humic acid, wet humic acid,urea, soil wetting aid, or a penetrant. When applied in the field toplants, billions of the selectively bred bacteria operate to covert andbreakdown organic matter into a form of micronutrient for plant uptake.The microbial solution can be applied through spraying, wetting,dipping, misting, drenching, showering, fogging, soaking, dampening,drizzling, dousing and splashing.

The biodiversity of Bacillus group and beneficial traits of bacillusspecies are useful in plant protection. Bacillus genus is widely spreadin nature. Bacillus species such as B. Subtilus, B. Megaterium, B.Amyloliquefaciens, B. lichniformis are carefully selected, for theirspecific profile which contains beneficial traits for plant protectionand growth promotion that comprise the synthesis in broad-spectrum withactive metabolites and easily adaptation in various environmentconditions that benefits plant bacterial interaction and advantageous offormulation process.

As plants roots exudates and lysates attracts and stimulate microbialactivity in the root surrounding soil, the zhizosphere (chemical spacearound the roots) became highly populated. Beneficial Bacillus spp.strains can compete with other bacteria and fungi that could adverselyaffect crops. They can inhibit phytopathogenic attacks such as “BasalStem Rot, phytophthora, fusarium”, or induce host-plant defense systemagainst potential pathogenic attacks, stimulate plant growth, improvenutrient uptake, and reduce negative environment traits.

Beneficial traits with agricultural purpose in Bacillus Subtilis andrelated species are detailed next. The species of bacillus group,particularly B. Subtilus, B. Megaterium, B. Amyloliquefaciens, B.lichniformis are extremely importance in agriculture, as phytopathogenicantagonist or plant growth promoters. It is often referring as “PlantGrowth Promoting rhizobacteria” or PGPR. PGPR are naturally occurringsoil bacteria that have the ability to colonize the roots, and the highconcentration and the amount of bacteria artificially created (added) asdetailed above enhances the stimulation of plant growth by phytohormonesproduction or by releasing beneficial organic compounds.

Beside plant growth stimulation, Bacillus Subtilis and its relatedspecies strain are involved in plant protection against phyto-pathogenicattacks. They act directly against pathogens by producing extracellularlytic enzyme and secondary metabolites with inhibitory growth action orinterfere by quorum quenching to disturb cell-to-cell communication ofthe infectious expression in pathogenic bacteria. They could alsocompete with plant pathogen for the available nutrient and niche.Another important role is the reduction of the infection process byinducing defense response in the host plant.

Each single microbial series is separately cultivated in its designatedcultivation medium, and the optimal pH in the growing and reproductionof different microbial series also varies. Therefore, proper control andregulation of pH of the cultivation medium are provided in the course ofbacterial cultivation and fermentation. The microbial series acquiresenergy through aerobic respiration. However, the aerobic respirationgenerally has to rely upon only the oxygen dissolved in the cultivationmedium, i.e., the dissolved oxygen, and the containment of the dissolvedoxygen in the cultivation medium is not always provided in sufficientamount and will be soonest consumed by bacteria since oxygen isdifficult to get dissolved in water. Therefore, constant air supply tothe microbial series is provided without interruption in the course ofthe cultivation and fermentation of the microbial series. Compositionsof cultivation medium selected and the optimal growing environmentconditions for each microbial series are detailed as follows:

When the cultivation of each microbial series is saturated in itscultivation medium, a cross cultivation is followed. The compoundmicrobial preparation differs from a single bacteria species or a singlemicrobial product for soil modification. In some embodiments, themicrobial life activities from multiple preselected microbial series areprovided that are mutually coordinated and contained for crops or plantsto get the results of specific fertilizers; that is, multiplemicroorganisms are screened from the soil and selectively bred to becomecapable of improving nutrition of the crops, and then to providenitrogen, phosphor, and potassium fertilizers important to the growth ofthe plants in organic means by taking advantage of interaction amongcompound microbial preparations. Wherein, the nitrogen fixing seriesfixes nitrogen molecules in the nature to make it a nitrogen source formanufacturing fertilizers; the phosphoric acid releasing series unlocksand converts insolvable phosphates in the soil into phosphor, ferrous,and calcium fertilizers; the yeast group series makes it available inthe making of vitamins and growing hormones, and decomposes organics toimprove disease-resistant sufficiency of the plants; the photosyntheticbacteria series while being applied in manufacturing of glucose secretscarotenoid and eliminates toxic substances including hydrogen sulfideand ammonia; the actinomyces series secrets antibiotic substances at aconstant amount on long-term bases to inhibit diseases; and the growingfactors producing series also releases on long-term basic a given amountof growing hormones to promote roots, stalks and leaves of crops orplants to grow strong. In some embodiments, one or more of the abovedescribed series of microbials are used.

In the course of cross cultivation, each of those eight microbial seriesmaintains intrigue symbiosis and shared prosperity among one another byplaying a critical role with secretions of its own particular activeorganics. For example, the nitrogen fixing series converts the molecularnitrogen into ammoniac nitrogen and the resultant ammoniac nitrogen ispartially to be consumed by the nitrogen fixing series, the remainingammoniac nitrogen is synthesized into organic nitrogen to be consumed byother bacterial series; and the yeast group series may catalyzepolysaccharide into simple sugar including glucose to be consumed bylactobacillus to convert into alcohol. Centering on the photosyntheticbacteria series and the yeast group series as leading cores, eachmicrobial series supports activities of other microbial series with itssynthetic proficiency while taking advantage of those substancesproduced by other microbial series to constitute a commonwealth circle.However, behind the big chain of food that relies upon symbiosissubstances, a survival game of gigantic resistance and wipe out takesplace among one another due to different properties. In the environmentseeing violent stimulation, new endocrines are produced. What's moreimportant is that any strain of bacteria survived is practically the topselected one with reliable activities.

Depending on the locality, season, depth of soil, the present inventionproduces the proper strains of the microbial series. Those who arefamiliar with the art may apply on various series, e.g. coccus,bacillus, vibrio, or Spirillum; different demands of oxygen, e.g.,aerobic and/or anaerobic; different environmental requirements, e.g.,acidophilus, alkalophilus, psycho-, meso-, or thermophilic to come upwith a locality-specific compound microbial preparation and differentmicrobial series may be used to produce compound microbial preparationsin various applications, e.g., for fertilizer, pesticide, or promotiongrowth of flowers and fruits.

Spores and/or colonies that enrich soils and/or provide plant biologicalcontrol agents are employed in some embodiments. These include bacteriasuch as Bacillus species, e.g., Bacillus subtilis, Bacillus cereus,Bacillus penetrans, Bacillus licheniformis, and Bacillus megaterium;fungi such as Trichoderma, e.g., Trichoderma hamatum, Trichodermaharzianum, Trichoderma polysporum, Trichoderma konigii, Trichodermaviride; yeast such as Saccharomyces cerevisiae; and mixtures of these.Other examples are given hereafter.

FIG. 3 shows exemplary antifungal activity express by different Bacillusspp. Strains. FIG. 3A shows exemplary Bacillus spp. antagonisticactivity against fusarium solani; while FIG. 3B shows exemplary fungalcell wall degradation, cell lysis and cytoplasm bleeding due to Bacillusspp. extracellular enzymes.

FIG. 4 shows exemplary cellulolytic enzymes synthesized by thebiological control agent which can be involved in two plant defensemechanism against phyto-pathogenic fungi. Exemplary cellulase activityexposed on Luria Bertani medium supplement with carboxyl-methylcellulose, reveal a clear halo of CMC degradation, after two days ofBacillus spp. strains incubation.

In one embodiment called AGN, a natural microbial soil rejuvenation andenrichment provides microbials including enzymes, metabolites andbeneficial microbial biomass that aid in building soil structure. Inthis embodiment, the concentration of microbes can include thefollowing:

Bacillus amyloliquefaciens 5.85×10^7 cfu/ml

Bacillus lichniformis 1.80×10^7 cfu/ml

Bacillus pumilus 4.05×10^7 cfu/ml

Bacillus subtilis 6.30×10^7 cfu/ml

and the penetrant can be water with Polyloxy-(1,2-Ethanedily),alpha-(nonylphenyl)-omega-hydroxy or Alcohol Ethoxylate.

The colony-forming unit (CFU or cfu) is a measure of viable bacterial orfungal cells. CFU measures only viable cells. For convenience theresults are given as CFU/mL (colony-forming units per milliliter) forliquids, and CFU/g (colony-forming units per gram) for solids.

Humic Acid can be leonardite and water, and the penetrant can be waterwith Polyloxy-(1,2-Ethanedily), alpha-(nonylphenyl)-omega-hydroxy. HumicAcid provides the necessary amino acids and protein to support an activemicrobial population to support active and healthy plant growth.

Penetrants or non-ionic penetrants facilitate even water movement intothe soil both horizontally and vertically while maintaining a very lowvolatility. In some embodiments, the penetrants comprises a surfactant,which can be used together with heptonic acid, alkyl polyglycoside,water soluble polyacrylamides (PAMs), and/or polysiloxane emulsion. Insome embodiments, the penetrants are selected to maintain soil moisturelevel near to root zone of predetermined plants, prevent leaching ofnutrients, or both. Other surfactants can be used in variousembodiments, for example: Nonionic surfactants include agents such assorbitan monolaurate, sorbitan monopalmitate, sorbitan sesquioleate,sorbitan trioleate, polyoxyethylene sorbitan monolaurate,polyoxyethylene sorbitan monostearate, polyethylene glycol monooleate,polyethylene glycol alkylate, polyoxyethylene alkyl ether, polyglycoldiether, lauroyl diethanolamide, fatty acid iso-propanolamide, maltitolhydroxy fatty acid ether, alkylated polysaccharide, alkyl glucoside,sugar ester, oleophillic glycerol monostearate, self-emulsifiableglycerol monostearate, polyglycerol monostearate, polyglycerol alkylate,sorbitan monooleate, polyethylene glycol monostearate, polyoxyethylenesorbitan monooleate, polyoxyethylene cetyl ether, polyoxyethylenesterol, polyoxyethylene lanolin, polyoxyethylene bees wax, andpolyoxyethylene hydrogenated castor oil; and the like. Anionicsurfactants include agents such as sodium stearate, potassium palmitate,sodium cetyl sulfate, sodium lauryl phosphate, sodium polyoxyethylenelauryl sulfate, triethanolamine palmitate, polyoxyethylene sodium laurylphosphate, and sodium N-acyl glutamate; and the like. Cationicsurfactants include agents such as stearyl dimethylbenzyl ammoniumchloride, stearyl trimethyl ammonium chloride, benzalkonium chloride,and laurylamine oxide; and the like.

In one embodiment, the penetrant can be about 20% alcohol ethoxylate andabout 80% orange oil. The penetrant can have one or more high terpene(50% by weight or more) based oils, one or more stabilizers, one or morechelating agents, one or more preservatives, one or more acidic pHadjusters and one or more organic solvents.

Surfactants can be used. Nonionic surfactants include agents such assorbitan monolaurate, sorbitan monopalmitate, sorbitan sesquioleate,sorbitan trioleate, polyoxyethylene sorbitan monolaurate,polyoxyethylene sorbitan monostearate, polyethylene glycol monooleate,polyethylene glycol alkylate, polyoxyethylene alkyl ether, polyglycoldiether, lauroyl diethanolamide, fatty acid iso-propanolamide, maltitolhydroxy fatty acid ether, alkylated polysaccharide, alkyl glucoside,sugar ester, oleophillic glycerol monostearate, self-emulsifiableglycerol monostearate, polyglycerol monostearate, polyglycerol alkylate,sorbitan monooleate, polyethylene glycol monostearate, polyoxyethylenesorbitan monooleate, polyoxyethylene cetyl ether, polyoxyethylenesterol, polyoxyethylene lanolin, polyoxyethylene bees wax, andpolyoxyethylene hydrogenated castor oil; and the like. Anionicsurfactants include agents such as sodium stearate, potassium palmitate,sodium cetyl sulfate, sodium lauryl phosphate, sodium polyoxyethylenelauryl sulfate, triethanolamine palmitate, polyoxyethylene sodium laurylphosphate, and sodium N-acyl glutamate; and the like. Cationicsurfactants include agents such as stearyl dimethylbenzyl ammoniumchloride, stearyl trimethyl ammonium chloride, benzalkonium chloride,and laurylamine oxide; and the like. Amphoteric surfactants such asalkylaminoethyl glycine chloride and lecithin; and the like.

To deploy, field persons mix AGN with clean water and let it set for aminimum of 1 hour or maximum overnight (keep air flows after mixed withwater) and apply directly to moist soil as a pre-plant, post-plant orseasonal treatment. The solution can be applied to soil, seeds, andplants. In some embodiments, the solution is not mixed with any otherfertilizers or fungicides and deployment of such chemicals should waitat least 72 hours before or after treatment.

For tank mixing, in one embodiment, field personnel can mix 1 gallon (4quarts or 3.8 liters) of AGN with minimum 100 gallons up to 1000 gallonsof clean water in a clean tank and free of chemical. The solution can beapplied at a rate of 2 to 4 quarts per surface acre or 4 to 8 liters persurface hectare.

For injection irrigation or fertigation, after tank mixing, AGN can beapplied by dosage rate of 0.5 to 1 gallon (2 to 4 quarts) per surfaceacre (4 to 8 liters per surface hectare). For side-dress or starter, thesolution can be applied at a rate of 1 to 2 quarts per surface acre or 2to 4 liters per surface hectare. Preferably, the solution can bedispensed with:

-   -   Localized Drip or Trickle    -   Sprinkler, or    -   Contour Furrows

AGN includes Advanced Microbes for Soil Rejuvenation and creates abalanced soil environment for healthy plant growth which requires theability to fully access the soil particulates and enriching them withphytonutrients utilizing highly concentrated microorganisms and organicmaterials.

The application of AGN creates superior root systems which canefficiently assimilate nutrients and micronutrients in the soil,resulting in higher yields and better plant health for all types ofplants, crops, and trees and increases yields, soil-root-plant health,balance soil nutrients, penetrate and loosen clay soils, leach saltsfrom root zones, reduce harmful nematodes, increase nutrient andmicronutrient uptake as well as increase cathode ion transfer.

Any microbial spores and/or colonies can be preserved using methods andsolutions of some embodiments. Spores and/or colonies of beneficial soiland plant pathogen biological control microorganisms are preferred.Microorganisms that grow rapidly and colonize substrata in soil aftertreatment with compositions of the invention are particularly preferred.These include, but are not limited to bacteria, e.g., Bacillus speciessuch as Bacillus subtilis, Bacillus cereus, Bacillus penetrans, Bacilluslicheniformis, and Bacillus megaterium; fungi, e.g., Trichoderma speciessuch as Trichoderma hamatum, Trichoderma harzianum, Trichodermapolysporum, Trichoderma konigii, and Trichoderma viride; and yeastspecies such as Saccharomyces cerevisiae. As illustrated below, mixturesof microorganisms can also be preserved, and are preferred in manyembodiments. Examples are given hereafter.

In the practice of the system, spores or whole microorganisms, includingharvested and/or lyophilized microbial colonies containing spores, areadded to solutions. The solutions can be formulated for any userequiring viable microbial spores and/or colonies such as forfertilizers, composting, food products, and pharmaceutical compositions.Liquid fertilizers are preferred for soil enrichment purposes. Watermiscible dry powders and/or granules such as lyophilized preparations ofspores and/or colonies are preferred in many embodiments. The amount ofspores or microorganisms added to solutions of the invention is notfixed per se, and necessarily is dependent upon the degree of soiland/or plant remediation required, the number and identity ofmicroorganism species needed in the formulation, and the concentrationof other ingredients in the formulation. Preferred embodiments employspores and/or colonies in amounts effective to achieve recolonization ofthe soil by spray application of the composition. Typical embodimentscontain sufficient spores and/or colonies to deliver from about 1000 toabout 1,000,000 colony forming units (CFU) per square foot when thepreparation is delivered.

Preservative solutions of some embodiments are colloidal in nature,containing humic acid and/or other organic macromolecules. By“colloidal” is meant a state of matter which comprises either largemolecules, aggregations of smaller molecules, or a combination of thetwo. Some embodiments contain large molecules such as humic acid and/ormethylene urea compounds of varying chain length. The particles aresurrounded by different matter such that a dispersed phase is surroundedby an external phase. Both phases may be solid or liquid (and sometimesgaseous). One phase comprises water in most embodiments; typical rangesare from about 35% to about 58% by weight water in the totalcomposition, but some embodiments contain less than about 20% by weightwater in the total composition.

Microorganisms and/or their spores which can be preserved usingformulations of the invention further exhibit a number of desirablecharacteristics related to soil enrichment and improvement of soilquality described above, such as biological control of plant pathogens(already mentioned); enhancement and/or production of desirablephtyohormones, e.g., auxins, giberillins and cytokinins; andsolubilization of phosphates. Certain strains of Bacillus subtilis, forexample, inhibit N. Galligena that colonize apple branch scars ifapplied to trees after leaf fall. E. herbicola and Pseudomonas isolateshave been shown to partially control fire blight of pome fruit trees.Several Bacillus species produce antibiotics useful when sprayed as aleaf or needle application on tobacco, Douglas fir, and apple trees, andthe natural protection of leaves provided by the buffering capacity ofphylloplane microorganisms has been demonstrated. Azobacter, Rhizobium,Bacillus, Klebsiella, Azospirillium, Enterobacter, Serratia,Agrobacterium, Arthrobacter, Aerobacter, Actinomyces, Bacillus,Pseudomonas, and other bacteria stimulate growth, increase yield, andproduce other positive results by various mechanisms including enhancingnutrient uptake, increasing germination, enhancing seedling emergence,stimulating de novo biosynthesis, and the like, when applied to fieldsof various food plants.

The resulting solutions supply carbon-rich organic materials in abioavailable form for soils and plants together with nutrients that feedthe microorganisms as they multiply after application. Solutions of someembodiments provide an excellent food source for the germination ofspores and/or colonies when the solutions are applied to soil or water.It is a further advantage that preferred solutions contain a widevariety of naturally occurring metabolites that can be readily absorbedby the growing microorganisms and enhance seed germination, rootdevelopment, and growth of plants in the soil.

As summarized above, some embodiments are formulated with microorganismspores and/or cultures useful in the prevention, control and/or cure ofplant diseases, particularly those of fungal origin. Illustrativeexamples are provided hereafter. One embodiment, for example, maintainsthe viability of Bacillus subtilis GB03 (EPA Reg. No. 7501-144), abacteria recognized to colonize developing root systems, suppressingdisease organisms such as Fusarium, Rhizoctonia, Alternaria andAspergillus that attack root systems. Compositions of the invention canbe used to treat developed root systems as well as developing rootsystems. As the root system develops, grows, and functions, the bacteriagrow with the roots, extending protection throughout the growing season.As a result of this biological protection, a vigorous root system can beestablished and maintained by the plants.

In addition, B. subtilis GB03 has been shown to increase the amount ofnodulation by nitrogen-fixing bacteria when used on many legumes. Thisimprovement in nodulation is a result of a healthier root system,allowing more sites for nodules to form from naturally-occurringsoilborne nitrogen-fixing bacteria. Illustrative examples follow.

FIG. 5 shows an exemplary AGN enzyme profiles isolation standard. Soilbacteria in the genus Bacillus are well known for contributions toimproving soil structure, nutrient availability and as a competitiveexcluder to harmful pathogens. Bacillus lichniformis produces a varietyof extracellular enzymes that are associated with the cycling ofnutrients in nature, thus improve nutrient availability and nutrientuptake. Bacillus pumilus is an agricultural fungicide. Growth of thebacterium on plant roots prevents rhizoctonia and fusarium spores fromgerminating. These strains are heavily involved with inhibition ofopportunistic pathogens as well as improving nutrient availability andnutrient uptake. Bacillus subtilis does nitrogen fixing; produceinhibitory compounds that reduce the growth of harmful microorganism. Itinterfere with the germination of plant pathogen spores and theirattachment to host plants, acts as a prebiotic conditioning plants owndefense mechanisms prior to attack from potential pathogens. Bacillusamyloliquefaciens had anti fungal properties and help nitrogen fixingavailability. Bacillius megaterium is a plant growth-promotingrhizobacteria (PGPR) and phosphate solubilizing. It promotes theactivation of plant defense responses and secretion of plantgrowth-regulating substances such as auxins, cytokinins and bacterialvolatiles. Phytohormones are involved in the control of growth and inalmost every important developmental process in plants. Bacterialsecretion of phytohormones can impact root architecture byoverproduction of root hairs and lateral roots and subsequentlyincreased nutrient and water uptake, thus contributing to growth.

Example 1 (AGN)

Microbes:

Bacillus amyloliquefaciens at 5.85×107^7 cfu/ml

Bacillus lichniformis at 1.80×107^7 cfu/ml

Bacillus pumilus at 4.05×107^7 cfu/ml

Bacillus subtilis at 6.30×107^7 cfu/ml

Humic Acid: Leonardite and H2O

Nitrogen: Urea and H2O

Penetrant: Polyloxy-(1,2-Ethanedily), Alpha-(nonylphenyl)-omega-hydroxyand H2O

Example 2 (AGN LTE)

Microbes:

Bacillus amyloliquefaciens at 5.85×107^7 cfu/ml

Bacillus lichniformis at 1.80×107^7 cfu/ml

Bacillus pumilus at 4.05×107^7 cfu/ml

Bacillus subtilis at 6.30×107^7 cfu/ml

Humic Acid: Leonardite and H2O

The above description is for the purpose of illustrating and notlimiting the present invention, and teaching the person of ordinaryskill in the art how to practice the invention. It is not intended todetail all those obvious modifications and variations of it which willbecome apparent to the skilled worker upon reading the description. Itis intended, however, that all such obvious modifications and variationsbe included within the scope of the present invention as defined in theappended claims. The claims are meant to cover the claimed componentsand steps in any sequence which is effective to meet the objectivesthere intended, unless the context specifically indicates the contrary.

The patents, papers, and book excerpts cited above are herebyincorporated herein by reference in in their entireties.

What is claimed is:
 1. A method for enhancing soil, comprising:preparing a microbial solution with microbes, a growth medium, andwater; iteratively and selectively breeding generations of microbes formicrobial strain selection with predetermined microbial gene profiles toarrive at a predetermined microbial solution in a highly concentratedform of at least 1×10⁷ cfu/ml (colony-forming units per milliliter),wherein multiple single microbial series are separately cultivated andfollowed with cross cultivation among the microbial series in a specificsequence, and by-products produced by the crossly cultivated microbialseries are provided as a highly concentrated solution; adding humic acidwith amino acids and protein to the concentrated solution to promote anactive microbial population to support active and healthy plant growth;and storing the microbial solution in a container for application as asoil amendment that is applied to the soil.
 2. The method of claim 1,comprising selecting a member of Bacillus as the microbe and providing acarrier from one of: liquid, water, dry humic acid, wet humic acid,urea, or a penetrant.
 3. The method of claim 1, wherein the growthmedium comprises a carbon source.
 4. The method of claim 1, wherein thegrowth medium comprises sugar, molasses, or maltodextrin.
 5. The methodof claim 1, comprising mixing the solution with 1 part microbes, 10 partcarbon source, and 1000 parts water.
 6. The method of claim 1,comprising aerating the solution for at least 20 minutes before applyingto the soil.
 7. The method of claim 1, comprising selecting the microbefrom Bacillus (B.) acidiceler, B. acidicola, B. acidiproducens, B.acidocaldarius, B. acidoterrestrisr, B. aeolius, B. aerius, B.aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. akibai, B.alcalophilus, B. algicola, B. alginolyticus, B. alkalidiazotrophicus, B.alkalinitrilicus, B. alkalisediminis, B. alkalitelluris, B. altitudinis,B. alveayuensis, B. alvei, B. amyloliquefaciens, B. a. subsp.amyloliquefaciens, B. a. subsp. plantarum, B. amylolyticus, B.andreesenii, B. aneurinilyticus, B. anthracis, B. aquimaris, B. arenosi,B. arseniciselenatis, B. arsenicus, B. aurantiacus, B. arvi, B.aryabhattai, B. asahii, B. atrophaeus, B. axarquiensis, B. azotofixans,B. azotoformans, B. badius, B. barbaricus, B. bataviensis, B.beijingensis, B. benzoevorans, B. beringensis, B. berkeleyi, B.beveridgei, B. bogoriensis, B. boroniphilus, B. borstelensis, B. brevisMigula, B. butanolivorans, B. canaveralius, B. carboniphilus, B.cecembensis, B. cellulosilyticus, B. centrosporus, B. cereus, B.chagannorensis, B. chitinolyticus, B. chondroitinus, B. choshinensis, B.chungangensis, B. cibi, B. circulans, B. clarkii, B. clausii, B.coagulans, B. coahuilensis, B. cohnii, B. composti, B. curdlanolyticus,B. cycloheptanicus, B. cytotoxicus, B. daliensis, B. decisifrondis, B.decolorationis, B. deserti, B. dipsosauri, B. drentensis, B. edaphicus,B. ehimensis, B. eiseniae, B. enclensis, B. endophyticus, B.endoradicis, B. farraginis, B. fastidiosus, B. fengqiuensis, B. firmus,B. flexus, B. foraminis, B. fordii, B. formosus, B. fortis, B.fumarioli, B. funiculus, B. fusiformis, B. galactophilus, B.galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B.ginsengi, B. ginsengihumi, B. ginsengisoli, B. globisporus, B. g. subsp.globisporus, B. g. subsp. marinus, B. glucanolyticus, B. gordonae, B.gottheilii, B. graminis, B. halmapalus, B. haloalkaliphilus, B.halochares, B. halodenitrificans, B. halodurans, B. halophilus, B.halosaccharovorans, B. hemicellulosilyticus, B. hemicentroti, B.herbersteinensis, B. horikoshii, B. horneckiae, B. horti, B.huizhouensis, B. humi, B. hwajinpoensis, B. idriensis, B. indicus, B.infantis, B. infernus, B. insolitus, B. invictae, B. iranensis, B.isabeliae, B. isronensis, B. jeotgali, B. kaustophilus, B. kobensis, B.kochii, B. kokeshiiformis, B. koreensis, B. korlensis, B. kribbensis, B.krulwichiae, B. laevolacticus, B. larvae, B. laterosporus, B. lautus, B.lehensis, B. lentimorbus, B. lentus, B. licheniformis, B. ligniniphilus,B. litoralis, B. locisalis, B. luciferensis, B. luteolus, B. luteus, B.macauensis, B. macerans, B. macquariensis, B. macyae, B. malacitensis,B. mannanilyticus, B. marisflavi, B. marismortui, B. marmarensis, B.massiliensis, B. megaterium, B. mesonae, B. methanolicus, B.methylotrophicus, B. migulanus, B. mojavensis, B. mucilaginosus, B.muralis, B. murimartini, B. mycoides, B. naganoensis, B. nanhaiensis, B.nanhaiisediminis, B. nealsonii, B. neidei, B. neizhouensis, B.niabensis, B. niacini, B. novalis, B. oceanisediminis, B. odysseyi, B.okhensis, B. okuhidensis, B. oleronius, B. oryzaecorticis, B.oshimensis, B. pabuli, B. pakistanensis, B. pallidus, B. pallidus, B.panacisoli, B. panaciterrae, B. pantothenticus, B. parabrevis, B.paraflexus, B. pasteurii, B. patagoniensis, B. peoriae, B.persepolensis, B. persicus, B. pervagus, B. plakortidis, B.pocheonensis, B. polygoni, B. polymyxa, B. popilliae, B.pseudalcalophilus, B. pseudofirmus, B. pseudomycoides, B. psychrodurans,B. psychrophilus, B. psychrosaccharolyticus, B. psychrotolerans, B.pulvifaciens, B. pumilus, B. purgationiresistens, B. pycnus, B.qingdaonensis, B. qingshengii, B. reuszeri, B. rhizosphaerae, B. rigui,B. ruris, B. safensis, B. salarius, B. salexigens, B. saliphilus, B.schlegelii, B. sediminis, B. selenatarsenatis, B. selenitireducens, B.seohaeanensis, B. shacheensis, B. shackletonii, B. siamensis, B.silvestris, B. simplex, B. siralis, B. smithii, B. soli, B.solimangrovi, B. solisalsi, B. songklensis, B. sonorensis, B.sphaericus, B. sporothermodurans, B. stearothermophilus, B.stratosphericus, B. subterraneus, B. subtilis, B. s. subsp. inaquosorum,B. s. subsp. spizizenii, B. s. subsp. subtilis, B. taeanensis, B.tequilensis, B. thermantarcticus, B. thermoaerophilus, B.thermoamylovorans, B. thermocatenulatus, B. thermocloacae, B.thermocopriae, B. thermodenitrificans, B. thermoglucosidasius, B.thermolactis, B. thermoleovorans, B. thermophilus, B. thermoruber, B.thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis,B. tianshenii, B. trypoxylicola, B. tusciae, B. validus, B.vallismortis, B. vedderi, B. velezensis, B. vietnamensis, B. vireti, B.vulcani, B. wakoensis, B. weihenstephanensis, B. xiamenensis, B.xiaoxiensis, and B. zhanjiangensis.
 8. The method of claim 1,comprising: applying enzymes, metabolites and microbial biomass to aidin building soil structure; and applying penetrants to facilitate evenwater movement into the soil both horizontally and vertically whilemaintaining low volatility.
 9. The method of claim 1, comprising mixingthe microbial solution with water and setting the solution for at leastone hour and flowing air after mixing with water; applying the setsolution directly to moist soil as a pre-plant, post-plant or seasonaltreatment; and applying fertilizers or fungicides after delaying atleast 72 hours before or after soil treatment.
 10. The method of claim1, wherein the microbes comprise Microbes Bacillus amyloliquefaciens at5.85×10⁷ cfu/ml, Bacillus lichniformis at 1.80×10⁷ cfu/ml, Bacilluspumilus at 4.05×10⁷ cfu/ml, or Bacillus subtilis at 6.30×10⁷ cfu/ml.